[目的]为增强不结球白菜对芜菁花叶病素养(TuMV)的抗性,研究探讨了不结球白菜中BcCHC1蛋白与TuMV之间的相互作用机制.[方法]首先从不结球白菜中鉴定出重链网格蛋白(CHC)基因家族的成员BcCHCs,随后克隆了 1个代表性的CHC1基因,并对其进行了亚细胞定位分析.通过双分子荧光互补实验(BiFC)筛选出与BcCHC1蛋白相互作用的TuMV蛋白,并利用病毒诱导的基因沉默技术(VIGS)来沉默BcCHC1基因,以评估其功能.[结果](1)成功克隆了不结球白菜的BcCHC1基因,该基因的编码序列长5 124碱基对,编码1 708个氨基酸.(2)在TuMV感染不结球白菜30 d后,实时荧光定量PCR分析显示,感染TuMV的不结球白菜中BcCHC1基因的表达量显著降低.(3)亚细胞定位研究表明,BcCHC1蛋白主要定位于烟草表皮细胞的细胞膜和细胞核.(4)BiFC实验进一步证实,BcCHC1蛋白能够与TuMV的CI和6K2蛋白发生相互作用,其中与CI的互作主要发生在细胞核内,而与6K2的互作则主要位于细胞膜.(5)通过对BcCHC1基因沉默株系的观察发现,沉默Bc-CHC1基因会导致植株死亡.[结论]BcCHC1蛋白通过与TuMV的CI和6K2蛋白相互作用,影响网格蛋白依赖的内吞作用途径及病毒复制,从而调控TuMV对不结球白菜的侵染.然而,BcCHC1基因调控TuMV侵染不结球白菜的具体机制仍需进一步研究.

Positive-sense single-stranded RNA(+ssRNA)viruses,the most abundant viruses of eukaryotes in nature,require the synthesis of negative-sense RNA(-RNA)using their genomic(positive-sense)RNA(+RNA)as a template for replication.Based on current evidence,viral proteins are translated via viral+RNAs,whereas-RNA is considered to be a viral replication intermediate without coding capacity.Here,we report that plant and animal+ssRNA viruses contain small open reading frames(ORFs)in their-RNA(reverse ORFs[rORFs]).Using turnip mosaic virus(TuMV)as a model for plant+ssRNA viruses,we demonstrate that small proteins encoded by rORFs display specific subcellular localizations,and confirm the presence of rORF2 in infected cells through mass spectrometry analysis.The protein encoded by TuMV rORF2 forms punctuate granules that are localized in the perinuclear region and co-localized with viral replication complexes.The rORF2 protein can directly interact with the viral RNA-dependent RNA polymerase,and mutation of rORF2 completely abolishes virus infection,whereas ectopic expression of rORF2 rescues the mutant virus.Furthermore,we show that several rORFs in the-RNA of severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)have the ability to suppress type Ⅰ interferon production and facilitate the infection of ve-sicular stomatitis virus.In addition,we provide evidence that TuMV might utilize internal ribosome entry sites to translate these small rORFs.Taken together,these findings indicate that the-RNA of+ssRNA vi-ruses can also have the coding capacity and that small proteins encoded therein play critical roles in viral infection,revealing a viral proteome larger than previously thought.

白菜类作物主要包括大白菜(Brassica rapa subsp.pekinensis)、不结球白菜(B.rapa subsp.chinensis)和芜菁(B.rapasubsp.rapa)等,可用作蔬菜、油料及饲料.白菜类作物在生产过程中常受到病毒病的危害,其中芜菁花叶病毒(TuMV)是主要病原物.近年来,在TuMV抗性基因的遗传分析、定位克隆及分子机制方面取得了一定研究进展,为培育优质抗病新品种奠定了基础.该文对国内外关于白菜类作物抗TuMV的遗传分析、抗性基因与分子标记、生物信息学与组学分析以及基因工程技术在抗TuMV研究中的应用进行综述,以期为白菜类作物抗TuMV研究和技术利用提供科学依据.